Phosphorylation of XPD drives its mitotic role independently of its DNA repair and transcription functions

The helicase XPD is known as a key subunit of the DNA repair/transcription factor TFIIH. However, here, we report that XPD, independently to other TFIIH subunits, can localize with the motor kinesin Eg5 to mitotic spindles and the midbodies of human cells. The XPD/Eg5 partnership is promoted upon phosphorylation of Eg5/T926 by the kinase CDK1, and conversely, it is reduced once Eg5/S1033 is phosphorylated by NEK6, a mitotic kinase that also targets XPD at T425. The phosphorylation of XPD does not affect its DNA repair and transcription functions, but it is required for Eg5 localization, checkpoint activation, and chromosome segregation in mitosis. In XPD-mutated cells derived from a patient with xeroderma pigmentosum, the phosphomimetic form XPD/T425D or even the nonphosphorylatable form Eg5/S1033A specifically restores mitotic chromosome segregation errors. These results thus highlight the phospho-dependent mitotic function of XPD and reveal how mitotic defects might contribute to XPD-related disorders.


Fig. S1. Immunolocalization of XPD and partners in XPD/WT cells.
(A) Localization of XPD, p44, Cyclin H and MMS19 during cytokinesis. XPD/WT cells were synchronized by double thymidine block, released in mitosis and analyzed by confocal immunofluorescence microscopy. Cells in telophase were identified according to their DAPI staining. The arrows point to the localization of XPD at the midbody Scale bar is 5µm. (B) Localization of XPD and MMS19 during metaphase. XPD/WT cells were synchronized by double thymidine block, released in mitosis and analyzed by confocal immunofluorescence microscopy. Cells in metaphase were identified according to their DAPI staining. Scale bar is 5µm. (C) Schematic representation of entire 760-aa XPD protein and the truncated form corresponding to the C-terminal part of XPD (444-760). Immunoprecipitated Eg5 (IP Eg5) was incubated with either entire Flag-XPD or Flag-XPD (444-760) fragment (as indicated, +). After washes, the coimmunoprecipitated proteins were resolved by SDS-PAGE and blotted with anti-Flag and Eg5. The results are representative of two independent experiments. (D) Recombinant Eg5 and XPD were incubated in the presence of single-strand DNA (ssDNA, 7.5nM) and immunoprecipitated with anti-Eg5 (IP Eg5). After washes, coimmunoprecipitated proteins were resolved by SDS-PAGE and blotted with anti-Eg5 and -XPD. The results are representative of two independent experiments.

Fig. S3. Immunolocalization of XPD and Eg5 in XPD/R683W cells.
(A) Immunofluorescence of XPD/R683W cells synchronized with double thymidine block and collected 9h after release. XPD and Eg5 localization was monitored through all the cell cycle stages (cells from each mitotic phase were identified according to their DAPI staining). The arrows point to irregular nuclear shapes and chromosome segregation errors. Scale bar is 5µm. (B) Immunoprecipitation of either XPD (IP XPD-GFP) or Eg5 (IP Eg5) from cells overexpressing (when indicated, +) C-terminally GFP-tagged-XPD. After washes, the co-immunoprecipitated proteins were resolved by SDS-PAGE and blotted with antibodies targeting XPD and Eg5.

Figure S5
A

Fig. S5. CREST and BubR1 in XPD/WT and XPD/R683W cells in prometaphase.
(A) Wild-type and XPD mutated cells were synchronized in prometaphase with Taxol (16h, 1µM). Chromosomes were stained with DAPI. The arrows point to XPD/R683W anaphase-like cells that escaped prometaphase arrest in the presence of chromosome segregation errors. Scale bar is 5µm. (B) Unmerged images for Flag-Tag, CREST and BubR1, from which merges presented Fig. 6E were made.
(C) XPD/WT and XPD/R683W cells overexpressing either XPD/WT, XPD/R683W, XPD/R683W-T425A or XPD/R683W-T425D were treated 8h with t-RA (5µM) and relative RARβ2 gene expression has been measured by RT-PCR. The mRNA levels were normalized to the GAPDH RNA amount. The RARβ2 mRNA expression is presented as n-fold induction relative to non-treated cells. The results represent the mean of two independent experiments performed in triplicates. Bars 1-3 correspond to the values presented Fig. 8G.
(D) Biotinylated AdMLP bound to streptavidin magnetic beads was incubated with RNAPII, TFIIA,-B,-D (TBP),-E, and -F, in the presence of core-IIH, CAK, XPD and Eg5 as indicated at the top of the panel. After washes, the sequential binding of different factors (RPB2, TFIIEβ, XPB, XPD, CDK7, Eg5) in the PIC formation, was evaluated by immunoblotting.